DESCRIPTION: Understanding the principles and mechanics of how biological structures are assembled from their macromolecular components is a challenging problem. Bacterial viruses are important models for these studies because they are highly tractable experimentally, and because they offer a large variety of informative features in their assembly pathways. Dr. Hendrix has used lambdoid phages to obtain fundamental insights into the mechanism of morphogenesis of the virus head and tail structures. The central emphasis of this continuation proposal is to understand the ways in which one protein can influence or determine how another protein folds or assembles, and more generally, how protein-protein interactions determine assembly pathways. Toward these goals, the project will examine in detail the assembly pathways and their structural correlates for phage HK97 heads and lambda tails and tail fibers. Through a combination of biochemical, genetic and structural experiments, the proposal will first of all attempt to refine the current understanding of the HK97 head assembly pathway by identifying and characterizing the intermediates in the pathway and investigating mechanisms in the initial assembly of the capsid shell. Second, the covalent cross-linking reactions between HK97 capsid protein subunits that Dr. Hendrix discovered recently will be characterized in terms of the biochemical mechanism, associated structural transitions, topology in the head structure and the role in the assembly process. A large collection of capsid protein mutants will be isolated to facilitate these studies, and possibly to reveal unforeseen features of the assembly pathway. Structural transitions in the capsid protein leading to the cross-linking reaction during the final stages of head assembly will be examined by a combination of genetics, cryo- electronmicroscopy and X-ray crystallography. Third, the proposed experiments will also examine the relationship between protein structure and biological function in the newly discovered "long tail fibers" of phage lambda, and characterize the role of two conserved, essential proteins in tail fiber assembly that are themselves not part of the mature tail structure. It is expected that the basic principles which emerge from these pathways would be directly applicable to the assembly mechanisms in other viruses, including medically relevant viruses, and more generally to the mechanisms of assembly of any protein-containing biological structure.